There has been considerable interest in developing methods of attaching various diagnostic and therapeutic agents to targeting proteins such as antibodies. Recent efforts include the conjugation of therapeutic agents, such as cytotoxic or antineoplastic drugs, to specific antibodies, such as monoclonal antibodies, to produce conjugates which can selectively target tumor cells while sparing normal tissues.
A large number of different classes of therapeutic agents have been considered, including beta-, gamma-, and alpha-emitting radioisotopes; plant and bacterial toxins; and a variety of antineoplastic drugs, including intercalating agents, antimetabolites, alkylating agents, and antibiotics. It is desirable to conjugate chemotherapeutic drugs to targeting molecules such as antibodies for the following reasons:
1. It has recently been shown that up to 1,000-fold more drug can be delivered to tumor cells when conjugated to an antigen-specific monoclonal antibody than is possible by the addition of free drug.
2. Pleiotropic drug resistance may arise following treatment with one of a number of chemotherapeutic drugs, resulting in inducing resistance to drugs of several classes. The mechanism(s) of this resistance are not entirely known, but it is known that this resistance can be partially overcome by antibody targeting of drugs.
3. Even though current chemotherapeutic drugs are active against only some of the major tumor types, the response rate in drug-insensitive tumor types may be increased by antibody-mediated delivery.
4. Many dose-limiting toxicities which are now seen with chemotherapeutic drugs can be reduced by conjugation to an antibody. A decrease in toxicity with concomitantly at least equal efficacy would provide a superior product with a higher therapeutic index.
To create a conjugate with a therapeutic agent and an antibody, the therapeutic agent may be directly linked to the antibody through nucleophilic substitution of certain groups on the antibody (e.g., amino, carboxyl, or sulfhydryl) or the drug may be conjugated to the antibody via a hetero- or homobifunctional cross-linker.
The linking group generally is heterobifunctional, having two different functionalities, one of which reacts with the drug and the other with the antibody. Linking groups may be small or quite long. For example, a relatively small linking group is carbonyl diimidazole. Large proteins or polymers ("carriers") have also been used as linking groups and offer the advantage of being able to bind many drug molecules to a single antibody molecule. Examples of -large proteins or polymers are poly-L-lysine, polyglutamate, dextran, and albumin, all of which have molecular weights in excess of 5000 daltons. These carriers generally are derivatized with small linking groups to bind drugs. See, for example, U.S. Pat. Nos. 4,699,784 and 4,046,722.
Drug conjugation to a protein or an antibody targeting molecule has generally been through covalent binding of the drug to the antibody directly or by covalently binding the drug molecule to the linking group. (Blair et al., J. Immunol. Meth. 59:129-44, 1983.) Even when the drug is linked to a carrier molecule such as albumin or dextran, the drug undergoes a modification to allow for the covalent conjugation of the drug. The drug modification often results in the loss of some of the activity of the drug molecule due to chemical modifications of some of the functional groups within the drug molecule.
In the case of some drug molecules, exposure to derivatization conditions may completely inactivate the drug. For other drug molecules, the derivatization may not be completely specific for groups intended for linkage but may also modify groups important for drug activity.
In contrast to such "drug-antibody" administration procedures, pretargeting approaches may be used to achieve therapeutic or diagnostic goals. Pretargeting approaches involve the interaction of two members of a high affinity binding pair such as a ligand-anti-ligand binding pair. Pretargeting is characterized by an uncoupling of the kinetics of the targeting moiety capable of localizing to a target site in vivo and the diagnostic or therapeutic active agent. This decoupling of the relatively slow localization kinetics of targeting moiety localization from the faster clearing active agent affords lower non-target exposure.
Accordingly, there exists a need in the field of drug conjugation to be able to attach multiple drug molecules to the targeting antibody, ligand or anti-ligand without covalent modification of the drug and loss of drug activity.